Tracking peripheral vascular function for six months in young adults following SARS‐CoV‐2 infection

Abstract SARS‐CoV‐2 infection is known to instigate a range of physiologic perturbations, including vascular dysfunction. However, little work has concluded how long these effects may last, especially among young adults with mild symptoms. To determine potential recovery from acute vascular dysfunction in young adults (8 M/8F, 21 ± 1 yr, 23.5 ± 3.1 kg⋅m−2), we longitudinally tracked brachial artery flow‐mediated dilation (FMD) and reactive hyperemia (RH) in the arm and hyperemic response to passive limb movement (PLM) in the leg, with Doppler ultrasound, as well as circulating biomarkers of inflammation (interleukin‐6, C‐reactive protein), oxidative stress (thiobarbituric acid reactive substances, protein carbonyl), antioxidant capacity (superoxide dismutase), and nitric oxide bioavailability (nitrite) monthly for a 6‐month period post‐SARS‐CoV‐2 infection. FMD, as a marker of macrovascular function, improved from month 1 (3.06 ± 1.39%) to month 6 (6.60 ± 2.07%; p < 0.001). FMD/Shear improved from month one (0.10 ± 0.06 AU) to month six (0.18 ± 0.70 AU; p = 0.002). RH in the arm and PLM in the leg, as markers of microvascular function, did not change during the 6 months (p > 0.05). Circulating markers of inflammation, oxidative stress, antioxidant capacity, and nitric oxide bioavailability did not change during the 6 months (p > 0.05). Together, these results suggest some improvements in macrovascular, but not microvascular function, over 6 months following SARS‐CoV‐2 infection. The data also suggest persistent ramifications for cardiovascular health among those recovering from mild illness and among young, otherwise healthy adults with SARS‐CoV‐2.


| INTRODUCTION
The novel coronavirus disease of 2019 (COVID-19) has been a major burden on worldwide healthcare and daily life for over two years. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has infected over 320 million and caused over 5.5 million deaths worldwide by January 2022 (Dong et al., 2020). SARS-CoV-2 can inhibit angiotensinconverting enzyme-2 (ACE2) receptor activity , as well as elicit systemic inflammation via a cytokine storm (Barnes et al., 2020;Paybast et al., 2020). Early investigations revealed SARS-CoV-2 is able to infect and cause inflammation of vascular endothelial cells and directly impact vascular function and blood flow regulation (Bhagat & Vallance, 1997;Hingorani et al., 2000;Varga et al., 2020), which can have significant systemic ramifications (Nakano et al., 2021;Paybast et al., 2020). This cascade of events may cause functional decrements seen throughout the vasculature, including vascular dysfunction among young (Nandadeva et al., 2021;Ratchford et al., 2020) and old adults (Ambrosino et al., 2021;Ergul et al., 2021;Lambadiari et al., 2021;Paneroni et al., 2021;Riou et al., 2021). Yet, to date, little is known regarding the long-term impact of the SARS-CoV-2 infection on the vasculature, especially among those with mild symptoms who may be overlooked or expected to not have long-term consequences.
While the severity of vascular consequences caused by SARS-CoV-2 infection is unclear, current studies focusing on vascular function have been cross-sectional in nature and have failed to establish a timeline following SARS-CoV-2 infection of vascular health and recovery among adults. Our group has previously observed significant vascular deficits acutely among young adults with SARS-CoV-2 , evidenced by decreases in brachial artery flow-mediated dilation (FMD), as an indication of macrovascular function that is highly correlated with coronary artery function (Thijssen et al., 2011(Thijssen et al., , 2019. In addition, a blunted reactive hyperemic (RH) response to single passive limb movement (sPLM), an indication of muscle microvascular dysfunction, was observed among these young adults 3 to 4 weeks after SARS-CoV-2 infection . These results were corroborated by others who identified a blunted hyperemic response to sPLM, in hospitalized individuals 2 to 4 months after SARS-CoV-2 infection (Paneroni et al., 2021). This blunted hyperemic response has even been observed during continuous PLM (cPLM) prior to SARS-CoV-2 symptom onset (Trinity et al., 2021). These results may suggest hypersensitive afferent and central hemodynamic responses to cPLM during acute infection and inflammatory responses such as during SARS-CoV-2 (Fudim et al., 2020). Further, lower FMD and RH in symptomatic young adults 1 to 4 months after infection with SARS-CoV-2 compared with healthy young adults (Nandadeva et al., 2021) may represent a prolonged disruption in vascular function. Likewise, middle-aged and older adults displayed reduced FMD compared with healthy controls at 2 (Ambrosino et al., 2021) and 3 (Riou et al., 2021) months following hospitalization, as well as at 4 months post-infection (Lambadiari et al., 2021). Despite growing evidence of vascular dysfunction among those infected with SARS-CoV-2, these findings lack a time-dependent association with symptom severity as individuals recover from SARS-CoV-2.
Thus, this investigation sought to extend our findings of vascular dysfunction in the 3 to 4 weeks following SARS-CoV-2 infection and examine the natural time course for potential vascular function improvements over a 6-month period. Using a series of non-invasive vascular assessments each month among young adults recovering from SARS-CoV-2, we hypothesized vascular function would improve from months one to six following SARS-CoV-2 infection (Carfi et al., 2020;Mahase, 2020), as evidenced by increases in macrovascular function in brachial artery FMD, microvascular function in brachial artery RH following cuff occlusion, and mobility muscle microvascular function in the femoral artery RH during PLM. As an exploratory analysis, we sought to study the afferent response via the central hemodynamic assessment to cPLM, which we hypothesized would be augmented from months one to six following SARS-CoV-2 infection. Additionally, we sought to explore if participants' functional outcome measures were cross-sectionally similar to healthy control participants by the completion of the study. Finally, this study sought to better understand the long-term effects of SARS-CoV-2 infection on circulating markers of inflammation, ROS, and NO bioavailability as potential mechanisms for vascular dysfunction and recovery. We hypothesized markers of inflammation (IL-6, CRP) and ROS (TBARS, protein carbonyl) would decrease, and antioxidant capacity (superoxide dismutase, SOD) and the potent vasodilator nitrite would increase from months one to six following SARS-CoV-2 infection. The current study provides a comprehensive, longitudinal assessment of peripheral vascular function and potential vascular mediators that has otherwise been lacking in the literature and may provide additional guidance for cardiovascular health and recovery efforts following SARS-CoV-2 infection in young adults.

| Participants
Otherwise healthy young adults who tested positive for SARS-CoV-2 using a nasopharyngeal swab polymerase chain reaction (PCR) assay reported to the laboratory 3-4 weeks following their positive test and for four additional visits approximately 1 month apart. All participants were non-smokers, free from chronic cardiovascular, metabolic, or renal disease, and female participants were premenopausal and not currently pregnant or breastfeeding. Likewise, these participants did not require hospitalization during or following infection.
As described previously , retrospective control participants were studied February 4-6, 2020 prior to the first confirmed case of COVID-19 in North Carolina, United States on March 3, 2020 (Services NDoHaH, 2020) and prior to the World Health Organization declaring COVID-19 a pandemic on March 11, 2020. Control subjects had not experienced flu-like symptoms.

| Study procedures
Testing took place in a quiet, environmentally controlled laboratory, with an ambient temperature of ~23°C. Participants returned for monthly testing to repeat the same ordered measurements. Participants arrived at the laboratory in a fasted state, having abstained from exercise, caffeine, and alcohol for at least 12 h before testing and were at least 4 h postprandial. Participants were asked to consume a low-fat diet and minimal food intake (Padilla et al., 2006;Vogel et al., 1997) when subjects could only abstain from food for 4 h instead of the traditional 6 h set forth by recommended guidelines (Harris et al., 2010;Thijssen et al., 2011) prior to testing. Upon arrival, participants were asked to rank their current symptom severity using an in-house survey, and anthropometric data were collected. Participants lay supine on a bed quietly for 20 min while being instrumented. Resting supine brachial artery blood pressures [systolic blood pressure (SBP); diastolic blood pressure (DBP); mean arterial pressure (MAP), as MAP = [((2/3)·DBP) + ((1/3)·SBP)]] and heart rate (HR) were obtained (Evolv, Omron Healthcare, Japan). Vascular function assessments were performed on participants. After vascular function testing, an antecubital venous blood draw was obtained. All Doppler ultrasound imaging and data processing and analysis were performed by a single, trained researcher throughout the study.

| SARS-CoV-2 symptom severity survey
Participants were asked to rank their SARS-CoV-2 symptoms each day of testing on a symptom severity survey (SSS), as previously described . On a scale of 0-100 of increasing severity, participants ranked their symptoms of chest pain, chills, diarrhea, dizziness or vertigo, dry cough, dry eyes, dry mouth, fatigue, fever over 37.9°C, headache, lack of appetite, loss of smell or taste (anosmia), muscle or body aches, nasal congestion or runny nose, nausea or vomiting, shortness of breath, difficulty breathing, dyspnea, sore joints, or sore throat. The values for all symptoms above a score of zero were totaled and averaged for total symptom severity. Mild symptoms were categorized as a symptom severity score of 0-33, moderate from 34 to 66, and severe as a score of 67 of 100. Additionally, participants subjectively provided their recent physical activity level each month based on days each week and duration of physical activity.

| Brachial artery flow-mediated dilation
Participants rested in the supine position for 20 min before the start of data collection with the right arm abducted 90 •, and the elbow joint extended at heart level. Brachial artery FMD measurements were obtained from the right brachial artery using current guidelines as a functional, upper limb marker of vascular function and cardiovascular risk (Thijssen et al., 2011). Baseline measurements of the right brachial artery diameter and blood velocity were taken for 1 min using a Doppler ultrasound system (GE Logiq eR7 and L4-12T-RS transducer, GE Medical Systems). Sample volume was optimized in relation to vessel diameter and centered within the vessel for each participant. Measurements of brachial artery diameter and velocity were obtained with the Doppler ultrasound in duplex mode with a B-mode imaging frequency of ~12 MHz and a Doppler frequency of approximately 4 MHz. An angle of intonation of ≤60° (Rizzo et al., 1990) was achieved for all measurements. Immediately after baseline measurements, a blood pressure cuff placed distal to the elbow, was rapidly inflated to 250 mmHg for 5 min. The blood pressure cuff was rapidly deflated, and brachial artery diameter and blood velocity were recorded for 2 min. Brachial artery diameter and blood velocity were analyzed offline for continuous second-by-second measurements (Cardiovascular Suite v. 4.0, Quipu). Brachial artery FMD was quantified as the maximal change in brachial artery diameter after cuff release, expressed as a percentage increase from pre-occlusion values (%FMD). Shear rate (SR) was calculated as follows: [SR (s −1 ) = blood velocity · 8/vessel diameter]. The %FMD was made relative to baseline diameter and SR to account for daily fluctuations in baseline diameter and SR-induced changes in vasodilation. Peak velocity and peak velocity relative to MAP were assessed to predict cardiovascular disease risk and future cardiovascular events (Huang et al., 2007).

| Brachial artery reactive hyperemia
Brachial artery RH was simultaneously measured and determined as the 2 min area under the curve (AUC) for the BF response following cuff occlusion, providing an index of microvascular function (Harris et al., 2010), which is inversely related to cardiovascular disease risk and predicts future cardiovascular events in healthy and diseased populations (Huang et al., 2007). Cumulative values of the AUC for SR and BF were integrated via the trapezoid rule and calculated as: Σ{yi [x(I + 1) -xi] + (½)[y(I + 1) -yi][x(I + 1) -xi]}; (x is time, y is shear rate, xi is initial time point, yi is initial blood velocity; Huang et al., 2007). Blood flow (BF) was determined as: BF, ml·min −1 = [blood velocity · π · (arterial diameter · 2 −1 ) 2 ] · 60. Brachial artery BF was made relative to MAP (vascular conductance, VC) to account for daily changes in driving pressure. In pilot testing, the average coefficient of variation for 2 individuals in triplicate assessment for baseline diameter (0.7%), peak diameter (0.5%), sum of shear at peak (9.4%), %FMD (10.7%), FMD/Shear (6.8%), and RH (2.1%) was similar to previously published reproducibility assessments for FMD (Ghiadoni et al., 2012).

| Femoral artery single passive leg movement
Femoral artery single PLM (sPLM) measurements were obtained from the right common femoral artery using current guidelines as a functional, lowerlimb assessment of microvascular function (Gifford & Richardson, 2017). While in the supine position with the participant's left leg supported on a table and right leg supported by a research team member at heart level, baseline measurements of the common femoral artery diameter and blood velocity, at least 3 cm proximal to the femoral artery bifurcation, were recorded for 1 min before performing the PLM maneuver using similar Doppler ultrasound system settings used for the brachial artery FMD procedure (B-mode imaging frequency of ~12 MHz and Doppler frequency of ~4 MHz). Immediately following baseline measurements, the research team member supporting the right thigh and ankle manually moved the knee joint one time through a 90° range of motion, flexion extension, at 1 Hz while common femoral artery diameter and blood velocity were recorded for 1 min after the movement. Common femoral artery diameter and blood velocity were analyzed offline for second-by-second measurements as described above (Cardiovascular Suite v. 4.0, Quipu).

| Femoral artery continuous passive leg movement
Following the sPLM, a cPLM assessment was performed on the same leg. The research team member manually moved the knee joint through a 90° range of motion, flexion extension, at 1 Hz continuously for 1 min, while common femoral artery diameter and blood velocity were recorded during this cPLM and 1 min AUC. Analysis was performed offline for continuous secondby-second measurements (Cardiovascular Suite v. 4.0, Quipu). The 1 min AUC response and VC were determined as indices of microvascular function (Gifford & Richardson, 2017). Heart rate (HR), stroke volume (SV), cardiac output (CO), and mean arterial blood pressure (MAP) were determined noninvasively using photoplethysmography (Finometer, Finapres Medical Systems BV) throughout the cPLM test to determine central hemodynamic responses to the afferent feedback elicited by the cPLM (Ives et al., 2016). Baseline, peak, change from baseline to peak (ΔPeak), and 60-s AUC (AUC60) were determined for all variables of interest. In pilot testing, the average coefficient of variation for 3 individuals in triplicate assessments for baseline blood flow, peak blood flow, delta blood flow, and area under the curve for sPLM (2.6%, 3.8%, 18.0%, and 18.7%, respectively) and cPLM (7.3%, 12.2%, 29.6%, and 22.2%, respectively) was similar to recently published reproducibility assessments of cPLM (Groot et al., 2022;Lew et al., 2021).

| Markers of oxidative stress, inflammation, nitric oxide bioavailability, and antioxidant levels
Venous blood draws were performed by a trained phlebotomist following vascular measurements. Plasma and serum were separated by centrifugation and stored at −80°C until analysis. Biochemical assays were performed according to the manufacturer's instructions for the oxidative stress biomarkers: TBARS (Cayman Chemical, Ann Arbor, MI, 10009055), as an indicator of MDA and lipid peroxidation, and serum protein carbonyl using a colorimetric assay (Cayman Chemical, Ann Arbor, MI, 10005020), as an indicator of protein oxidation; inflammatory biomarkers, plasma CRP (Cayman Chemical, Ann Arbor, MI, 10011236) and plasma IL-6 using enzyme-linked immunosorbent assays (Cayman Chemical, Ann Arbor, MI, 501030); the serum antioxidant biomarker SOD using an activity assay (Cayman Chemical, Ann Arbor, MI, 706002) and NO bioavailability, through plasma measurement of the putative endpoint, nitrite, utilizing a NO assay kit (ThermoFisher Scientific, Vienna, Austria, EMSNO).

| Statistical analyses
Statistical analyses were performed using commercially available software (IBM SPSS Statistics Version 26, Armonk, NY; SAS Version 9.4, Cary, NC). Each continuous variable was checked for normality using Kolmogorov-Smirnov tests. Normality was confirmed visually with QQ-plot observations. Linear mixed models were used to determine the main effects of time (monthly visits) for all outcome variables. Studentized residuals falling outside of three standard deviations would have been considered outliers and removed from analysis; however, no outliers were detected. Significant findings were determined by an alpha level of 0.05 for pairwise comparisons. Eta-squared was measured as an indicator of the main effect size for FMD, RH, sPLM, and cPLM functional outcome measures. Tukey-Kramer post hoc correction was used where significant effects were observed, and adjusted p-values are reported. Hedge's g was measured using pooled standard deviation for all the groups along with a correction factor, as an indicator of effect size for FMD, RH, sPLM, and cPLM functional outcome measures. As an exploratory analysis and to provide the context of expected healthy control data, one-way repeated measures ANOVA with planned comparison analyses were performed between the SARS-CoV-2 participant visits and healthy control participants to determine group differences for FMD, RH, sPLM, and cPLM functional outcome measures. Pearson correlations were performed to determine the potential associations between the changes to functional indices of vascular function (i.e., FMD, FMD/ Shear, sPLM BF AUC, or cPLM BF AUC) and the changes to circulating biomarkers (i.e., CRP, IL-6, SOD, TBARS, protein carbonyl, and nitrite) from months one to six; these correlations may exist on an individual subject level even when group difference do not exist from month to month. For these correlations, each participant with a M1 and M6 visit was plotted as an individual data point. Values are expressed as means ± SD.
Participant visit compliance was 78% with 12 of the 16 participants completing at least 5 visits. Two participants (1 M/1F) did not complete the M3-6 study visits; one other participant (1F) did not complete the M3 study visit; one other participant (1F) did not complete the M4-6 study visits; one other participant (1F) did not complete M3 or M5 study visits; one other participant (1F) did not complete M5-6 study visits; three other participants (3 M) did not complete M6 study visit; two other participants (1 M/1F) did not complete M5 study visit. Due to high attrition during M5, the decision was made to collapse the last subject visits from M5 (3M/0F) and M6 (4M/5F) to create a final study visit. The final testing visit (7 M/5F, 174 ± 15d) ranged from 143-197d post-positive PCR test result, indicated by "M6" in the results to indicate a final study visit.

| SARS-CoV-2 symptom severity survey
Most participants (n = 15) had mild, lingering symptoms from M1 to M4, while one female participant was consistently asymptomatic from M1 to M6. Symptom severity at each visit for those participants who experienced symptoms (SSS >0) can be found in Table 1. Participants were devoid of any medication usage other than six female subjects using oral contraceptives. Four subjects (3 M/1F) received either one or both doses of the SARS-CoV-2 vaccine during the study (three Moderna and one Pfizer). One female participant received her first dose of Moderna between M4 and M6. Two male participants received their first dose between M2 and M3, and the second dose between M4 and M5 (one Moderna and one Pfizer). One other male participant received his first dose of Moderna between M1 and M2, and the second dose between M2 and M3. Vaccinated subjects did not exhibit noticeable differences in measured outcomes compared with the unvaccinated subjects in the current investigation. Further, participants' study visits were scheduled at least 2 weeks following participants' previous vaccine dates to diminish the impact of vaccination effects on outcome variables, a design previously utilized in vaccination studies (Lind et al., 2012). These vaccinated participants' results were generally similar to all other unvaccinated participants' results. Therefore, participants were not separated by vaccination status.

| Reactive hyperemia
Brachial artery RH results are displayed in Table 2.

| Markers of oxidative stress, inflammation, nitric oxide bioavailability, and antioxidant levels
Measurements of oxidative stress, inflammation, nitric oxide (NO) bioavailability, and antioxidant levels are presented in Table 4. Due to the amount of adequate blood samples available, 8-14 participant samples were available for each biomarker, as indicated in Table 4. Blood samples were analyzed for every study visit for all biomarkers, except protein carbonyl, which was only analyzed for M1, M3, and M6 study visits. There was no main effect of time for protein carbonyl (p = 0.329) across time. Additionally, there was no main effect for time for TBARS (p = 0.424), IL-6 (p = 0.207), CRP (p = 0.368), nitrite (p = 0.942), and SOD (p = 0.334) across time. The individual participant's slopes (7 M/5F) from M1 to M6 for %FMD were not associated with the individual participant slopes (7 M/5F) from M1 to M6 for CRP (R = 0.024, p = 0.940), IL-6 F I G U R E 2 Brachial artery reactive hyperemia expressed as area under the curve (AUC) (a) and made relative to mean arterial pressure (AUC·mmHg −1 ) (b) during recovery from SARS-CoV-2. A repeated-measures ANOVA (p < 0.05; time, 5 levels) was performed to compare RH responses to a 5-min cuff occlusion between month 1 (n = 8 M/8F), month 2 (n = 8 M/8F), month 3 (n = 7 M/5F), month 4 (n = 7 M/6F), and month 6 (n = 7 M/5F). Individual male (circle) and female (triangle) participant data points are denoted. Data are means ± SD.

| DISCUSSION
The purpose of this study was to determine the natural time course for potential recovery from acute vascular dysfunction by tracking vascular function in the arms and legs, as well as various biomarkers, for a 6-month period post-SARS-CoV-2 infection. In support of our hypothesis, %FMD increased throughout the 6-month period, and was significantly greater at the six-month time point compared with the study entry, indicating improvements in conduit vascular function in the brachial artery. Brachial artery F I G U R E 3 Single passive limb movement. Common femoral artery blood flow, represented by area under the curve (AUC) at 60 s during recovery from SARS-CoV-2 and retrospective healthy control participants (a). A repeated-measures ANOVA (p < 0.05; time, 5 levels) was performed to compare BF responses between month 1 (n = 8 M/8F), month 2 (n = 8 M/8F), month 3 (n = 7 M/5F), month 4 (n = 7 M/6F), and month 6 (n = 7 M/5F). Femoral artery blood flow during month 6 for participants with SARS-CoV-2 and retrospective health control participants (b). Two-tailed student's t-tests for two samples of equal variance were performed between control (n = 5 M/15F) and SARS-CoV-2 at month 6 (n = 7 M/5F) participants. Individual male (circle) and female (triangle) participant data points are denoted (a). ‡ p < 0.05, group effect. Data are means ± SD. RH, a measure of microvascular function, was unaltered 6 months following SARS-CoV-2 infection. Likewise, the hyperemic response to the mobility muscle microvascular assessments sPLM and cPLM were unaltered 6 months following SARS-CoV-2 infection, despite a lower sPLM AUC60 BF for young adults recovering from SARS-CoV-2 compared to healthy retrospective control subjects acutely after infection. Interestingly, there were no differences in circulating biomarker levels throughout the 6-month testing period, nor did the slope over the 6-month period for the circulating biomarkers correlate with the functional vascular biomarkers. However, the results from the FMD and sPLM measurements provide evidence for impaired vascular function following SARS-CoV-2 infection. Together, these data suggest persistent consequences to cardiovascular health among those recovering from SARS-CoV-2, even with mild illness and in young, otherwise healthy adults.

| Brachial artery flow-mediated dilation
Brachial artery FMD is an assessment of macrovascular function (Thijssen et al., 2011) which correlates with coronary artery vascular function and is predictive of cardiovascular disease risk (Broxterman et al., 2019). SARS-CoV-2 is known to induce a systemic inflammatory response (Paybast et al., 2020). Previous studies demonstrate there is also a direct inflammatory response in endothelial cells (Varga et al., 2020), which could lead to vascular dysfunction and directly impact BF regulation (Bhagat & Vallance, 1997;Hingorani et al., 2000;Zimmer et al., 2011). From our initial findings , the acute decrease in %FMD among young adults with SARS-CoV-2 (2.7%) cross-sectionally compared to healthy young adults (8.8%) is concerning, as F I G U R E 4 Continuous passive limb movement. Common femoral artery blood flow, represented by area under the curve (AUC) at 60 s (a) and vascular conductance at 60 s, represented by AUC (b) during recovery from SARS-CoV-2. A repeated-measures ANOVA (p < 0.05; time, 5 levels) was performed to compare BF responses between month 1 (n = 8 M/8F), month 2 (n = 8 M/8F), month 3 (n = 7 M/5F), month 4 (n = 7 M/6F) and month 6 (n = 7 M/5F). Individual male (circle) and female (triangle) participant data points are denoted (a). Data are means ± SD. this measurement is associated with a higher risk for cardiovascular events, such as heart attack, stroke, or death (Yeboah et al., 2009). As mentioned, other groups have attempted to provide evidence of the potentially longlasting effects of SARS-CoV-2 on vascular function among hospitalized older adults (Ambrosino et al., 2021) which may be related to disease severity (Riou et al., 2021) and last for several months (Lambadiari et al., 2021). Similar to the aforementioned studies in older adults, an additional investigation in otherwise healthy young adults recovering from SARS-CoV-2 tested at a single time point, between 1 and 5 months after infection, providing further evidence of a symptom-dependent, negative association with vascular function as indicated by %FMD (Nandadeva et al., 2021). However, this study did not investigate when this vascular improvement occurs during recovery, with measurements being taken once per participant, and at various time points across 4-21 weeks post-infection. This current investigation sought to bridge this gap and provide longitudinal data on younger individuals with mild or no symptoms. We provide measurements at multiple time points to indicate %FMD at peak dilation improved in young adults by 6 months after SARS-CoV-2 infection which remains lower than healthy controls in the current investigation ( Figure 1). Similar findings among crosssectional investigations have revealed these decrements in macrovascular function may persist for at least 12 months following SARS-CoV-2 infection (Faria et al., 2022;Ikonomidis et al., 2022;Zota et al., 2022), suggesting longterm vascular decrements. SARS-CoV-2 elicits the cytokine storm which can cause an inflammatory state (Paybast et al., 2020) and elevate resting sympathetic levels . Our group also investigated the effects of SARS-CoV-2 on the autonomic function in young healthy individuals during the acute phase of infection . These results indicate mildly higher sympathetic activity in those infected, compared with a healthy control group. Previous studies show that an elevated sympathetic drive can impede the FMD response (Hijmering et al., 2002;Thijssen et al., 2006). Likewise, a lower heart-rate-variability in individuals with a SARS-CoV-2 infection, but displaying mild symptoms, compared with healthy, control counterparts (Kurtoglu et al., 2022;Solinski et al., 2022). Furthermore, persistent sympathetic overactivation (66% higher MSNA) along with blunted vascular function (45% lower FMD) have been observed cross-sectionally among older adults who were hospitalized with COVID-19 and were studied 1 year after infection compared to well-matched control subjects (Faria et al., 2022), further suggesting long-lasting autonomic dysfunction among those previously infected with COVID-19. With these studies in mind, the initial reduction in %FMD may be attributed to autonomic dysregulation. However, the autonomic function should be investigated in long-term recovery in individuals similar to this participant pool to further support that sympathetic activity is in fact impeding the FMD response.

| Reactive hyperemia
RH is used as a measurement of microvascular function. Unlike FMD, RH response to cuff occlusion and release is minimally influenced by NO (Engelke et al., 1996). The lower %FMD and normal RH response observed acutely  along with the increases seen with %FMD and lack of change in RH after several months following infection in our study may indicate SARS-CoV-2 may preferentially affect NO bioavailability and macrovascular function more than microvascular function among these young mild cases of SARS-CoV-2. In our previous study, the group of young adults 3 to 4 weeks after contracting SARS-CoV-2 had similar RH values compared with the healthy controls . Tehrani et al. found that severe SARS-CoV-2 patients who required mechanical ventilation displayed impaired microvascular function and particularly lower endothelial function, which persisted for 3 months post-infection (Tehrani & Gille-Johnson, 2021), indicating long-term microvascular decrements in severe cases. The previous investigation by Nandadeva et al. observed a symptom-dependent association with microvascular function in young adults with SARS-CoV-2 as well (Nandadeva et al., 2021), with a lack of difference between the COVID-19 group and the control group. However, when divided into COVID-19 subgroups based on symptom presence, the symptomatic group had lower RH values compared with the asymptomatic and control groups, while the asymptomatic participants' FMD values did not differ from the control group. The lack of change in RH observed in the current investigation, nor any cross-sectional difference at Month 6 compared to healthy controls (Figure 2), could be attributed to the participant population displaying mild symptoms and may represent a divergence in vascular dysfunction and recovery throughout the vascular tree.

| Femoral artery single passive limb movement
PLM is a novel, non-invasive assessment of mobility muscle microvascular function and has been observed to be greater than 80% dependent on NO bioavailability (Gifford & Richardson, 2017;Trinity et al., 2010). Those with decreased cardiovascular health, such as elderly individuals and those with heart failure tend to have a blunted PLMinduced hyperemic response (Francisco et al., 2021;Gifford & Richardson, 2017). The use of the sPLM, as opposed to the cPLM, evokes peripheral hemodynamic responses while minimizing central hemodynamic responses, provoking NO-dependent microvascular vasodilation (Ives et al., 2016;Venturelli et al., 2017). Our recent observation, utilizing a preliminary cohort of subjects, of an acute decline in the hyperemic response to sPLM among young adults 3-4 weeks after SARS-CoV-2 infection compared with healthy controls , indicates a reduced ability of the small arterioles to dilate when provoked by physical distortion. These results align with those observed in the elderly 2 to 4 months after hospitalization for SARS-CoV-2 infection (Paneroni et al., 2021). At rest, the elderly cohort displayed similar femoral artery diameter, but a significantly lower blood velocity and femoral BF (~100 ml/min) compared with a healthy control group. Additionally, an ~150 ml/min lower peak femoral BF, ~125 ml/min lower ΔBFpeak and lower AUC was observed in the COVID group compared with control after the sPLM movement (Paneroni et al., 2021). Interestingly, the current investigation observed no improvements in the hyperemic response of the sPLM over the course of 6 months. Differential responses in vascular function and structure between the arms and the legs, during both rest and exercise, have been noted by our group Ratchford et al., 2020) and others (Newcomer et al., 2004(Newcomer et al., , 2005Parker et al., 2006;Richardson et al., 2006). Further, there is growing evidence that FMD is not primarily NO-mediated (Green et al., 2014;Wray et al., 2013), which could represent divergent mechanisms between the findings of improved FMD and a lack of change in either sPLM or NO in these 6 months following SARS-CoV-2 infection. Given the similar leg BF responses between participants with SARS-CoV-2 6 months after infection and healthy controls to sPLM ( Figure 3) and cPLM (Figure 4), perhaps limb-dependent (arm versus leg) and/or other cellular mechanisms (NO versus non-NO-mediated vasodilation) may be responsible for these limb-specific and macro-versus microvascular findings. Yet, more work is certainly needed to further examine the vascular recovery rates across the upper and lower limbs among those recovering from SARS-CoV-2, quite possibly using additional methodologies to discern NO and non-NO-mediated dilatory mechanisms.

| Femoral artery continuous passive limb movement
SARS-CoV-2 infection can cause an increase in mitochondrial reactive oxygen species (ROS), evoking increases in oxidative stress and inflammation as well as decreases in bioavailability of the potent endothelial-derived vasodilator, NO (Fedorova et al., 2014;Morris et al., 2020;Pearce et al., 2020;Saeed & Mancia, 2021). Endothelialderived NO production has been noted to play a crucial role in the change in vascular tone during PLM (Gifford & Richardson, 2017). The movement of the leg elicits endothelial NO release and other dilatory mechanisms, resulting in rapid dilation of the vascular bed. The vascular decrements caused by SARS-CoV-2 may contribute to blunted exercising BF  and whole-body aerobic capacity (Acar et al., 2021), ultimately impacting quality of life among SARS-CoV-2 survivors. The current investigation does not show significant improvements in measures of microvascular function, indicated by BF and VC responses both before and during 60 seconds of cPLM, which may further corroborate the notion of time and regional differences in vascular recovery throughout the vascular tree.
Inflammation that develops in return of an active immune response may have sweeping implications through systemic circulation (Guo et al., 2020). The pro-inflammatory mediators released in the cytokine storm have the ability to cross the blood-brain barrier and increase the activation of the sympathetic nervous system (Pongratz & Straub, 2014), as evidenced in the acute phase of SARS-CoV-2 infection . This increase in sympathetic activity can have detrimental effects on several physiologic systems, including alterations in cardiac contraction (Moreira et al., 2017), and impairments in vascular function (Thijssen et al., 2006). Further, autonomic deficits have been described among patients with SARS, 6 months after infection (Lo et al., 2005), providing further evidence for long-lasting autonomic impairments among those infected with coronaviruses. While peripheral hemodynamic changes were not observed over time, there were significant changes in some central hemodynamic measures. A significant increase in SV AUC 60 was observed at M4, whereas, ΔpeakCO did not statistically decrease over the 6 months. These results may indicate the central hemodynamics were overcompensating for the decreased vascular function to produce a similar peripheral hyperemic response to cPLM. Following months of recovery, this central hemodynamic compensation may not be needed. Likewise, muscle afferents, stimulated during the cPLM, can increase HR, SV, and CO (Venturelli et al., 2014(Venturelli et al., , 2017, as our current results indicate. These factors, in addition to the NO release, can contribute to the increased perfusion of the dilated vascular bed (Gifford & Richardson, 2017;Ives et al., 2016;Trinity et al., 2010). These alterations in autonomic and cardiovascular measures indicate a disruption in neurovascular control and can contribute to the change in central hemodynamic measures, and lack of vascular alterations. 4.5 | Markers of oxidative stress, inflammation, nitric oxide bioavailability, and antioxidant levels

| Oxidative Stress
The chronic effects of oxidative stress have implications for arterial health, NO bioavailability (Moncada et al., 1991), and vascular function (Eskurza et al., 2004). Elevated oxidative stress among individuals acutely infected by coronaviruses has become increasingly apparent and may be a primary contributor to SARS-CoV-2 complications and prolonged recovery (Delgado-Roche & Mesta, 2020;Suhail et al., 2020). Elevated MDA levels have been observed in many disease states including aged adults (Mutlu-Turkoglu et al., 2003), and have been observed to even be approximately six times higher in COVID patients 4 months post-infection compared with both hypertensive patients and a control group (Lambadiari et al., 2021), suggesting lipid peroxidation may persist for several months after infection. Interestingly, our currently reported TBARS levels across the first 6 months following SARS-CoV-2 infection in young adults appear to be approximately five times higher than our previously reported TBARS levels for young healthy adults , suggesting lipid peroxidation may persist for several months even among young adults with mild symptoms. Likewise, elevated protein carbonyl levels may be associated with numerous disease states (Dalle-Donne et al., 2003;Huang et al., 2013;Johnson & Travis, 1979;Totan et al., 2009) and with age (Gil et al., 2006;Mutlu-Turkoglu et al., 2003). In the current investigation, we did not observe a change to either protein carbonyl despite being ~6 times higher than our previously reported healthy young adults , which may be an indication of a lack of improvement in protein oxidative stress among young adults with mild symptoms of SARS-CoV-2 during the first 6 months following infection. While quarantine procedures prevented earlier observations than 3-4 weeks after SARS-CoV-2 infection in the current investigation, significant elevations in oxidative stress during the first weeks of infection may be even higher than the levels observed here, which has been theorized to be central to the acute symptoms caused by SARS-CoV-2 (Laforge et al., 2020). Regardless, these persistently higher levels of lipid and protein oxidative stress compared to previously reported healthy young adults may be deleterious for the peripheral vasculature and could increase the risk of cardiovascular disease progression (Siti et al., 2015) which should be considered even among those with mild symptoms after SARS-CoV-2 infection.

| Inflammation
Coronaviruses, such as SARS-CoV and SARS-CoV-2, provoke the production and release of pro-inflammatory cytokines (Barnes et al., 2020). Elevated IL-6 levels may be associated with adverse clinical outcomes (Coomes & Haghbayan, 2020;Kermali et al., 2020) including cardiovascular complications (Ridker et al., 2000), and may depend on disease severity Gao et al., 2020;Henry et al., 2020;Huang et al., 2020;McGonagle et al., 2020;Ponti et al., 2020;Ruan et al., 2020;Wu et al., 2020;Zhang et al., 2020;Zhou et al., 2020). A meta-analysis of IL-6 concentrations in hospital patients with COVID-19 revealed levels approximately three times higher in patients with more severe COVID-19 complications, compared with patients suffering from other, "noncomplicated," diseases (Coomes & Haghbayan, 2020). Furthermore, persistent inflammation, noted by high concentrations of several circulating cytokines, is seen in patients with COVID-19 through 2 months post-hospital discharge (Montefusco et al., 2021). However, more work is surely needed to confirm these preliminary observations. Similar to IL-6, CRP is a strong indicator of the presence and severity of COVID-19 infection Kermali et al., 2020), as well as a strong predictor for the risk of cardiovascular events (Avan et al., 2018;Ridker et al., 2000) and mortality (Ruan et al., 2020). CRP concentrations have been shown to be significantly elevated in the initial stages of infection in hospitalized COVID-19 patients (Ali, 2020;Mandal et al., 2021) as well as at hospital discharge, but they have been observed to return to healthy levels 4 to 6 weeks following hospital discharge (Mandal et al., 2021). This current investigation observed consistent, low concentrations of CRP both acutely, and throughout recovery compared to our previously reported in healthy young adults . Given the complexity of the inflammatory cascade, observational timing is critical to observe changes in the multitude of inflammatory mediators. Recently, IL-10, an anti-inflammatory mediator which may act in a negative feedback loop within the inflammatory cascade and also correlates with disease severity , was observed to be significantly elevated only days after SARS-CoV-2 infection but prior to any patient symptoms (Trinity et al., 2021), suggesting the timing of the inflammatory cascade may vary between subjects and with varying disease severity.

| Antioxidants/nitric oxide bioavailability
Reactive oxygen species (ROS) play various roles in regulating vascular function (Wolin, 2009). Excess production of ROS can cause a decrease in NO bioavailability, resulting in endothelial damage and subsequent vascular dysfunction (Suematsu et al., 2002;Wolin, 2009), vasoconstriction, inflammation, and redox imbalance (Beltran-Garcia et al., 2020). Surprisingly, circulating SOD levels have been observed to be elevated in a disease-severity manner among those hospitalized with COVID-19 (Mehri et al., 2021), which may be a remedial mechanism to counteract the effects of high oxidative stress. Interestingly, our results of consistent levels of SOD over time may be intended to defend against consistently high oxidative stress levels. It has been speculated antioxidant capacity may be more impacted during the early phases of infection (Laforge et al., 2020). Therefore, future investigations should focus on both acute as well as longer term impacts on antioxidant capacity. Further, NO has been shown to be negatively impacted by SARS-CoV-2 infection, regardless of disease severity, with individuals showing reduced nitrite levels 4 months after hospitalized treatment and discharge (Wang et al., 2021). Indeed, NO has been shown to exert inhibitory effects on SARS-CoV-2 (Stefano et al., 2020) and may even be a therapeutic target for those recovering from SARS-CoV-2, as clinical trials of inhaled NO underway (Ignarro, 2020). While the current investigation provides evidence of consistent nitrite levels months after SARS-CoV-2 infection among young adults with a mild symptom, there will continue to be an urgent need to gauge the long-term implications of alterations or lack thereof in NO bioavailability on peripheral vascular function as it pertains to lasting health decrements caused by SARS-CoV-2 (Marshall, 2020).

| Limitations
We recognize several limitations to this real-world, observational investigations, including the use of oral contraceptives in most of our female participants (n = 6). Although contraceptive use was consistent throughout the testing period, it could impact vascular function (Friedman et al., 2011). Despite the diurnal variation of vascular function remaining contentious (Bau et al., 2008;Jarvisalo et al., 2006;Kim et al., 2015), subjects were encouraged and primarily attended similar testing times across visits, although this was not always possible, and some visit start times may have varied ~4 h throughout the day. Although vascular function alterations within the menstrual cycle may be minimal (Williams et al., 2020), female participants reported to the lab at 1-month intervals which minimized menstrual cycle fluctuations in vascular function within participants, while the cycle phase between participants was not controlled for. Our participant pool consisted of otherwise healthy, young adults, free of any cardiovascular and pulmonary diseases, and exhibited noto-mild symptoms from the infection. Advancing age and the presence of chronic medical conditions are widely known to be associated with the disease severity of COVID-19 infection (Jordan et al., 2020;Zhou et al., 2020). Those with preexisting comorbidities have more severe symptoms and higher mortality (Figliozzi et al., 2020). A limitation of this real-world data is the inability to collect data prior to SARS-CoV-2 infection. Further, a time control group was not possible during this early, fluid stage of the COVID-19 pandemic . Many SARS-CoV-2 cases are undetected, and more than half of patients may have an initial false negative PCR test (Arevalo-Rodriguez et al., 2020), making it difficult to determine if control participants have been infected with the SARS-CoV-2 virus before or especially during longitudinal testing. Vascular alterations may even predate positive SARS-CoV-2 testing (Trinity et al., 2021), making longitudinal testing of a control group inappropriate during an active pandemic. While this investigation was the first to longitudinally investigate the impact of SARS-CoV-2 in the proceeding months after infection, more work is needed across a broad range of populations and among those with more severe illnesses. Certainly, future studies can learn from these initial findings when choosing time points, patient populations, and sample sizes.

| CONCLUSION
This study was the first to longitudinally track peripheral vascular function in the arms and legs as well as the circulating biomarkers are known to regulate vascular function in young adults recovering from SARS-CoV-2 for 6 months after initial infection. Among mild cases of SARS-CoV-2 in young adults, the vascular function required several months to recover, which was unrelated to circulating markers of inflammation, oxidative stress, antioxidant capacity, and NO bioavailability. Together, these results suggest persistent ramifications for cardiovascular health, even among those recovering from mild illness and among otherwise healthy, young adults with SARS-CoV-2 which should be critically considered as the impact of SARS-CoV-2 in the general population continues. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.